ShRNA靶向沉默VTCN1基因对鼻咽癌细胞的影响

发布时间：2018-06-25 05:12

本文选题：鼻咽癌 + RNA干扰　；参考：《临床耳鼻咽喉头颈外科杂志》2017年10期

【摘要】：目的:通过研究VTCN1在鼻咽癌细胞中的表达情况,采用慢病毒载体靶向沉默VTCN1基因体外对人鼻咽癌细胞的抑制作用。方法:通过RT-PCR技术和蛋白质免疫印迹法,检测人鼻咽癌细胞株CNE1,HONE-1,NP65和HNE2的VTCN1表达情况,筛选出VTCN1表达量最多的细胞株,进行后续的实验。用脂质体lipofectamine2000将沉默VTCN1的慢病毒载体转染高表达VTCN1的HNE2细胞,筛选出稳定细胞株;采用RT-PCR技术和蛋白质免疫印迹法检测VTCN1ShRNA对VTCN1基因的靶向沉默效率;ckk-8、基质胶侵袭实验检测VTCN1沉默后HNE2细胞增殖和侵袭的影响;蛋白质免疫印迹实验检测VTCN1沉默后,JAK/STAT通路主要蛋白的磷酸化水平的变化。结果:VTCN1靶向沉默的细胞VTCN1表达受到显著抑制;RT-PCR和蛋白质免疫印迹法检测发现稳定转染ShRNA后,VTCN1的mRNA表达水平和蛋白表达水平均明显下降(P0.05);转染VTCN1ShRNA后HNE2细胞的增殖和侵袭能力均明显降低(P0.05);VTCN1沉默后,JAK、STAT磷酸化水平明显下降(P0.05)。结论:VTCN1ShRNA能有效沉默VTCN1的表达,显著抑制HNE2细胞的增殖和侵袭,推测其与下调JAK/STAT信号通路蛋白的活化相关。
[Abstract]:Aim: to investigate the expression of VTCN1 in nasopharyngeal carcinoma cells and to investigate the inhibitory effect of lentivirus vector targeting VTCN1 gene on human nasopharyngeal carcinoma cells in vitro. Methods: the expression of VTCN1 in human nasopharyngeal carcinoma cell line CNE1HONE-1nNP65 and HNE2 was detected by RT-PCR and Western blotting. The lentivirus vector silencing VTCN1 was transfected into HNE2 cells with high expression of VTCN1 by liposome lipofectamine2000, and stable cell lines were selected. RT-PCR and Western blot were used to detect the effect of VTCN1ShRNA on the targeting silencing efficiency of VTCN1 gene and the effect of VTCN1ShRNA on the proliferation and invasion of HNE2 cells after VTCN1 silencing. Western blot assay was used to detect the phosphorylation of major proteins in the JAK / stat pathway after VTCN1 silencing. Results the expression of VTCN1 was significantly inhibited in the cells targeted to VTCN1. RT-PCR and Western blot analysis showed that the mRNA expression level and protein expression level of VTCN1 decreased significantly after stable transfection of ShRNA (P0.05), the proliferation of HNE2 cells and the expression of VTCN1 protein in HNE2 cells were observed after transfection of VTCN1 ShRNA. The invasive ability was significantly decreased (P0.05) and the phosphorylation level of JAKN STAT was significantly decreased after VTCN1 silencing (P0.05). Conclusion the cell proliferation and invasion of HNE2 cells can be significantly inhibited by the inhibition of VTCN1 expression by 1: VTCN1ShRNA, which may be related to the down-regulation of JAK / stat signaling pathway protein activation.
【作者单位】：武汉大学中南医院耳鼻咽喉头颈外科;武汉大学人民医院耳鼻咽喉科;
【分类号】：R762

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